The epithelial cells lining the intestinal mucosa separate the underlying tissue from components of the intestinal lumen. Innate immunity mediated by intestinal epithelial cells (IECs) provides rapid protective functions against microorganisms. Innate immunity also participates in orchestrating adaptive immunity. Key components in innate defence are defensins. To study the production of defensins and how it is affected by intestinal inflammation IECs were isolated from the small and large intestines of patients suffering from ulcerative colitis (UC), Crohn´s disease (MbC), celiac disease (CD), and from controls, and analyzed by quantitative RT-PCR (qRT-PCR) and immunoflow cytometry. Defensin expressing cells were also studied by in situ hybridization and immunohistochemistry. Normally, only small intestinal Paneth cells express human α-defensin 5 (HD-5) and HD-6. In UC colon IECs, HD-5, HD-6, and lysozyme mRNAs were expressed at high levels. In Crohn´s colitis colon the levels of HD-5 and lysozyme mRNAs were also increased although not to the same extent as in UC. No increase was detected in MbC with ileal localization. Metaplastic Paneth cell differentiation in UC colon was primarily responsible for the expression of the antimicrobial components. Human β-defensin 1 (hBD-1) mRNA was more abundant in large than in small intestine of controls, and remained unchanged in UC and MbC. hBD-2 mRNA was barely detectable in normal intestine and was induced in UC IECs but not in MbC IECs. mRNAs for the recently discovered hBD-3 and hBD-4, were detected in IECs from both small and large intestine. Both hBD-3 and hBD-4 mRNA were significantly increased in IECs of UC patients but not of MbC patients. Bacteria and IL-1β induced hBD-2 but not hBD-1 mRNA in colon carcinoma cell lines. IFN-γ, but not TNF-α or IL-1β, augmented hBD-3 expression in these cells, while none of the agents induced hBD-4. High antimicrobial activity of IECs in UC may be a consequence of changes in the epithelial lining, which permit the adherence of microorganisms. Unexpectedly, in situ hybridization revealed expression of hBD-3 and hBD-4 mRNAs by numerous lamina propria cells in colonic tissue from UC patients. These cells were identified as plasma cells (CD138+). hBD-3 and hBD-4 mRNAs were also demonstrated in the plasmacytoma cell line U266. This is the first demonstration of defensins in plasma cells. The four prominent constituents of the intestinal glycocalyx, carcinoembryonic antigen (CEA), CEA cell adhesion molecule 1 (CEACAM1), CEACAM6 and CEACAM7 all seem to play a critical role in innate defence of the intestinal mucosa by trapping and expelling microorganisms at the epithelial surface. The inducibility of these molecules in colonic epithelial cell lines was analyzed by qRT-PCR, immunoflow cytometry, and immunoelectron microscopy. IFN-g but not bacteria, LPS, TNF-α, or IL-1β modified the expression of CEA, CEACAM1 and CEACAM6. None of these agents modified CEACAM7 expression. IFN-γ was shown to have two effects: a direct effect on CEACAM1 transcription, and promotion of cell differentiation resulting in increased CEA and CEACAM6 and decreased CEACAM7 expression. Scanning electron microscopy of jejunal biopsies from children with CD revealed the presence of rod shaped bacteria in ~40% of patients with active CD, but only in 2% of controls. 19% of treated CD patients still had adhering bacteria. Presence of bacteria is not due to lack of antimicrobial factors. In fact, HD-5, HD-6, and lysozyme mRNA levels were significantly increased in IECs of patients with active CD. hBD-1 and hBD-2 were unchanged. Lack of induction of hBD-2 may reflect disturbed signalling in IECs of CD patients. Analysis of CEA and CEACAM1 mRNA/protein expression showed no differences between CD patients and controls. Analysis of the mucins MUC2 and MUC3 revealed significantly increased MUC2 levels in active disease and unchanged MUC3. Immunohistochemistry demonstrated goblet cell metaplasia as well as staining of the apical portion of absorptive cells. Glycosylation status of proteins was studied by lectin histochemistry. Goblet cells in the mucosa of CD patients were stained by the lectin UEAI. This was not seen in controls. The lectin PNA stained the glycocalyx of controls but not that of CD patients. Thus, unique carbohydrate structures of the glycocalyx/mucous layer are likely discriminating features of CD patients and may allow bacterial binding. We conclude that the intestinal epithelium is heavily involved in the innate defence of the mucosa and that its reactive pattern is affected by intestinal inflammation. Keywords: human intestinal mucosa; epithelial cells; innate immunity; defensin; ulcerative colitis; Crohn´s disease; celiac disease; glycoαcalyx; mucin

No description available.

Pretendeu-se, no presente estudo, verificar a segurança e a eficácia do transplante de células-tronco derivadas do tecido adiposo (ASC) em ratos com UC induzida por ácido trinitrobenzenosulfonico (TNBS). A população celular foi isolada do tecido adiposo por dissociação mecânica e cultivada. O comportamento celular foi verificado por meio da morfologia, proliferação, viabilidade, capacidade de aderência à superfície plástica e parâmetros de diferenciação osteogênica, condrogênica e adipogênica. Os animais foram avaliados, considerando-se os aspectos clínicos, macroscópicos, microscópicos e bioquímicos. No modelo experimental de UC, a infusão de ASC reduziu significativamente a presença de aderências entre o cólon e órgãos adjacentes, bem como diminuiu a quantidade de células inflamatórias na mucosa lesada. Conclui-se que as ASC podem promover e/ou acelerar o processo de regeneração da mucosa intestinal inflamada e assim, serem empregadas como uma opção terapêutica com amplo potencial de aplicabilidade no tratamento da DII. / It was intended in this study verify the safety and efficacy of the transplantation of adipose derived stem cells (ASC) in rats with ulcerative colitis induced by trinitrobenzenosulfonico acid (TNBS). The cell population was isolated from the adipose tissue by mechanical dissociation and cultured. The cell behavior was verified by the morphology, proliferation, viability, ability to adhere to the plastic surface and parameters of osteogenic differentiation, adipogenic and chondrogenic. The animals were evaluated, considering the aspects clinical, macroscopic, microscopic and biochemical. In experimental ulcerative colitis, infusion of ASC significantly reduced the presence of adhesions between the colon and adjacent organs and decreased the amount of inflammatory cells in the injured mucosa. It is concluded that the ASC can promote and/or accelerate the healing process of the intestinal mucosa inflamed and thus be employed as a therapeutic option with wide potential application in the treatment of IBD.

No trato digestório a colite ulcerativa apresenta necrose no intestino como processos patofisiológicos. Este projeto visou estudar neurônios com códigos químicos do sistema nervoso entérico (SNE) e a morfologia estrutural do intestino grosso de animais com colite ulcerativa. Grupos: a) Colite: injetados com TNBS, b) PBS: injetados com PBS e c) controle. Os tecidos foram preparados por métodos imunohistoquímicos de duplas marcações do receptor P2X7 com NOS, ChAT, Calb, Calr, anti-HuC/D (pan-neuronal) e S100 (células glias). No grupo Colite, no plexo mioentérico, o receptor P2X7 estava diminuído. No tecido lesado apresentou aumento de neutrófilos e da lâmina própria, alteração de colágeno e destruição do epitélio e células caliciformes. Reduziram colocalizações de neurônios com receptor no plexo mioentérico e aumento no plexo submucoso. Houve reduções nas densidades e áreas dos neurônios no SNE. Conclui-se que a colite afetou os neurônios entéricos e células gliais, causou alterações morfológicas, sendo assim, pode afetar motilidade intestinal. / In the digestive tract ulcerative colitis have a bowel necrosis as pathophysiological processes. This project aimed to study neurons with their respective chemicals codes the enteric nervous system (ENS) as well as structural morphology of the distal colon of animals with ulcerative colitis. Groups: a) colitis: TNBS injected, b) PBS: PBS injected and c) control. The tissues were prepared by immunohistochemical methods for double marking with P2X7 receptor, ChAT, Calb, Calr, anti-HUC / D (pan-neuronal) and S100 (glial cells). In the colitis group, the myenteric plexus, the P2X7 receptor decreased. In the injured tissue showed increased neutrophils, alteration and destruction of collagen and epithelial goblet cells. There were reduced colocalizations of neurons with receptor in myenteric plexus and increase in submucosal plexus. There were reductions in the densities and areas neurons of ENS. Concluded that colitis affected enteric neurons and glial cells, causing morphological changes and could be affect intestinal motility.

Inflammatory bowel diseases (IBD) are an autoimmune illnesses affecting gastrointestinal tract. The main types include ulcerative colitis and Crohn's disease. Recently, primary sclerosing cholangitis (PSC) has also been associated with IBD. PSC is a chronic liver disease associated with bile duct stenosis. The exact pathogenesis and etiology of these diseases is not clear, despite the great efforts of the scientific community. They are multifactorial diseases that are associated with dysbiosis of intestinal microbiota. Their diagnosis is based on for patients unpleasant endoscopic examinations and therefore the search for new serum biomarkers is needed and appreciated target of scientific interest. In the first part of diploma thesis, we focused on the reactivity of peripheral blood cells of IBD patients to 10 selected representatives of typical intestinal microbiota: Lactobacillus plantarum, Bifidobacterium adolescentis, Blautia coccoides, Roseburia intestinalis, Eubacterium rectale, Faecalibacterium prausnitzii, Ruminococcus flavefaciens, Bacteroides thetaiotaomicron, Prevotella ruminicola and Escherichia coli. Reactivity of CD, UC and PSC- IBD patients was increased after stimulation with Faecalibacterium, Lactobacillus and Prevotella. However, we got low percentage of cytokine-producing cells,...

The intestinal mucosa is made up of complex secreted mucus layer consist of mainly mucin 2 (MUC2) and antimicrobial components that defend the underlining cellular barrier from intrusion by luminal microbiota and toxins. In inflammatory bowel diseases (IBD), the mucosal integrity is compromised. This can result from a combination of altered host genetics, gut immune responses and environment factors. However, it is the presence of intestinal bacteria that is central to the pathogenesis of IBD. As part of the dynamic gut microbial flora, mucolytic bacteria produce a wide range of glycosidases that are able to remove the outer oligosaccharide chains of MUC2, which allow other luminal bacteria to further degrade the mucin. We hypothesised that increased mucolytic bacteria will cause excessive degradation of the mucus layer, which in turn, allow more luminal bacteria to be in close proximity to the underlining epithelial cells resulting in inflammation. Consistent with our group’s previous semi-quantitative bacterial 16S rRNA gene clone library analysis, we found increased Ruminococcus gnavus in non-inflamed ulcerative colitis (UC) mucosa. R. gnavus was previously isolated by others based on its mucolytic property. In this study, we quantify total mucosa-associated bacteria and mucolytic bacteria, namely, R. gnavus, R. torques, Akkermansia muciniphila and bifidobacteria. We were able to show quantitatively that total mucosa-associated bacteria were increased in IBD. There was also a population shift in the mucosa-associated mucolytic bacteria, which were increased overall. There was significantly more R. gnavus in non-inflamed IBD biopsies. For the first time, we were also able to demonstrate that R. gnavus can degrade human MUC2 in vitro. To examine whether the numerical association of R. gnavus in IBD does have functional influence on intestinal inflammation and Paneth cell antimicrobial peptide gene expression, we fed mice with R. gnavus. Interestingly, R. gnavus feeding did not result in histological or molecular evidence of gut inflammation; however, it was able to specifically induce Paneth cell cryptdins and lysozyme P genes expression in 3 week old, antibiotic pre-treated C57BL/6 mice. This demonstrated that R. gnavus is not a pathogenic bacterium, which will directly cause colitis. However, the increased Paneth cell response suggested the need for host innate defence when R. gnavus is increased. Other than bacterial degradation, altered host genetics will also influence the mucus barrier. There is evidence to suggest that the MUC2 gene is highly unstable and is susceptible to gene copy number variation (CNV). Therefore, we hypothesised that MUC2 CNV is present, which may result in altered oligomerisation of the MUC2 glycoprotein causing endoplasmic reticulum stress of the goblet cells that appears to be characteristic of UC. Currently, our data partly support the presence of MUC2 CNV. However, further investigation is required to verify the MUC2 CNV identity. Only then can a high throughput methodology be designed to screen a large population for any association with IBD.

<p>The accumulation of eosinophil granulocytes (EOS) at sites of inflammation is a common feature of astma, allergic rhinitis and inflammatory bowel disease. The aim of the present investigation was to study the mechanisms involved in this accumulation.</p><p>Bronchoalveolar lavage (BAL) fluid obtained from patients with birch-pollen allergy lavaged during season exhibited increased eosinophil chemotactic activity compared with pre-season BAL fluid from the same patients. We identified IL-5, IL-8 and RANTES as the main eosinophil chemotactic agents in the BAL fluid. Only EOS from allergic donors responded to IL-8. IL-2 inhibited albumin-stimulated eosinophil migration towards buffer or chemoattractants. EOS from allergic subjects were less sensitive to this inhibition than EOS from normal subjects, and in vitro priming of the EOS with IL-5 prevented the inhibitory effect of IL-2. We therefore hypothesise that IL-2 acts as an autocrine regulator of EOS migration, and that this inhibitory effect may be down-regulated in allergy, resulting in increased migration of EOS towards chemotactic factors. The stimulation of eosinophil migration by albumin is mediated by PI3 kinase. Decreased expression of CD49d and CD49f caused by albumin may decrease the adhesiveness of the EOS, which in turn may facilitate migration. We found a higher chemotactic activity in perfusion fluids from patients with ulcerative colitis than from control patients. The chemotactic activity correlated with the concentrations of eosinophil granule proteins in the perfusion fluids. IL-5 and TNF-α were identified as two of the chemotactic agents in the perfusion fluid that were inhibited by steroid treatment. Agents with steroid-insensitive chemotactic activity remain to be identified.</p>

The accumulation of eosinophil granulocytes (EOS) at sites of inflammation is a common feature of astma, allergic rhinitis and inflammatory bowel disease. The aim of the present investigation was to study the mechanisms involved in this accumulation. Bronchoalveolar lavage (BAL) fluid obtained from patients with birch-pollen allergy lavaged during season exhibited increased eosinophil chemotactic activity compared with pre-season BAL fluid from the same patients. We identified IL-5, IL-8 and RANTES as the main eosinophil chemotactic agents in the BAL fluid. Only EOS from allergic donors responded to IL-8. IL-2 inhibited albumin-stimulated eosinophil migration towards buffer or chemoattractants. EOS from allergic subjects were less sensitive to this inhibition than EOS from normal subjects, and in vitro priming of the EOS with IL-5 prevented the inhibitory effect of IL-2. We therefore hypothesise that IL-2 acts as an autocrine regulator of EOS migration, and that this inhibitory effect may be down-regulated in allergy, resulting in increased migration of EOS towards chemotactic factors. The stimulation of eosinophil migration by albumin is mediated by PI3 kinase. Decreased expression of CD49d and CD49f caused by albumin may decrease the adhesiveness of the EOS, which in turn may facilitate migration. We found a higher chemotactic activity in perfusion fluids from patients with ulcerative colitis than from control patients. The chemotactic activity correlated with the concentrations of eosinophil granule proteins in the perfusion fluids. IL-5 and TNF-α were identified as two of the chemotactic agents in the perfusion fluid that were inhibited by steroid treatment. Agents with steroid-insensitive chemotactic activity remain to be identified.

Genetiniai ir funkciniai tyrimai parodė, kad oksidacinį stresą sukeliančios reaktyvios deguonies formos (angl. Reactive oxygen species, ROS) užima svarbų vaidmenį uždegiminių žarnų ligų (UŽL) patogenezėje. NADPH oksidazės šeimos fermentai (Nox1, Nox2, Nox3, Nox4, Nox5, Duox1 ir Duox2) - svarbus ROS šaltinis organizme. Šių fermentų gaminamos ROS molekulės veikia kaip antriniai signalų nešikliai, kurie aktyvina bei reguliuoja įvairius biologinius procesus, tokius kaip augimas, ląstelių diferenciacija, apoptozė ar uždegimas. NADPH oksidazės izoformos randamos gaubtinės žarnos ir virškinamojo trakto barjeriniuose audiniuose, turi tiesioginį kontaktą su žarnų simbiotinėmis bei patogeninėmis bakterijomis. Todėl manoma, kad šių fermentų biologinės funkcijos gali būti susijusios su įgimtais apsauginiais, gynybiniais mechanizmais, ląstelės signaliniais keliais bei virškinamojo trakto uždegiminių ligų patogeneze. Darbo tikslas – ištirti epitelio ląstelių NADPH oksidazės reikšmę žarnų uždegimo patogenezei, sergant opiniu kolitu ir DSS sukeltu kolitu. Mūsų darbas atskleidė NADPH oksidazės svarbą žarnų uždegimo patogenezei. Mes pirmieji parodėme, jog NADPH oksidazės fermentų slopinimas pasižymi uždegimą malšinančiu poveikiu pirminėms žmogaus ir pelių žarnų epitelio ląstelėms in vitro. / Genetic and functional studies indicated the importance of reactive oxygen species (ROS) induced oxidative stress in the development of chronic inflammatory bowel disease (IBD). NADPH oxidase enzymes (Nox1, Nox2, Nox3, Nox4, Nox5, Duox1 and Duox2) - are the important source of ROS in the organism. These enzymes - derived ROS act as intracellular second messengers for a variety of cellular receptor signal transduction pathways, and they play pivotal roles in various biological activities, including host defence, cell growth and differentiation, stimulation of pro-inflammatory genes, and cell death. The epithelial NADPH oxidase isoforms are highly expressed in the colon, particularly in the colon epithelial cells. These enzymes come into close contact with normal and pathogenic bacteria and may play an important role in local innate immune, cell signalling pathways and inflammation in the gut. The aim of this study is to investigate the role of NADPH oxidase of colon epithelial cells in the pathogenesis of colon inflammation during ulcerative colitis and DSS-induced colitis. The results of our study revealed the importance of NADPH oxidase in the pathogenesis of colon inflammation. Moreover, we are the first to show that the treatment with NADPH oxidase inhibitors has a protective effect against pro-inflammatory action of LPS in human and mice primary colon epithelial cells in vitro.

Clostridium difficile colitis (CDC) is associated with a higher risk of acute death among hospitalized ulcerative colitis (UC) patients. However, the risk of colectomy with CDC in these patients has varied across studies. No study has assessed the long-term health impact of CDC in UC patients. Therefore, the present study evaluated the impact of CDC on five-year health outcomes of hospitalized UC patients based on Ontario health administrative data. No overall association was observed between CDC and five-year risks of colectomy or death in overall cohort. However, patients who were discharged from hospital without undergoing colectomy demonstrated marginally higher five-year risks of colectomy and hospital re-admission. Mortality risk and length of stay during index hospitalization were also higher in patients with CDC. Analysis of a parallel cohort of UC patients derived using a published case definition corroborated most of these results, but demonstrated a higher five-year mortality risk with CDC.